Exquisitely selective turn-on probes of kinase activation and localization Protein kinase-mediated phosphorylation is a vital posttranslational modification in eukaryotic cell signaling. Efforts to understand these complex signaling pathways have been hampered by a lack of selective kinase inhibitors. Nearly all known kinase inhibitors bind in the highly conserved ATP pocket. Although potency is easy to obtain within the ATP-pocket, selectivity is difficult to obtain due to the similar nature of this binding pocket across the kinome. Non-ATP-competitive inhibitors usually possess higher degrees of selectivity than their ATP-competitive counterparts, however, they generally suffer from a lack of potency. In contrast, both high potency and high selectivity can be obtained with bisubstrate inhibitors. Bisubstrate kinase inhibitors interact with both binding pockets; interactions within the ATP pocket provide potency while interactions within the substrate site provide selectivity. Here, we propose to develop methodology for the modular construction of bisubstrate kinase inhibitors. Our goal is to develop this methodology in a way that can target any kinase and yield a potent and highly selective inhibitor. There are many potential applications of these proposed perfectly selective kinase inhibitors. Here, we propose to develop highly selective and cell permeable probes with `turn-on' fluorescence when bound to their target kinase. Kinase probes that target active kinase populations enables imaging applications in live cells and tissues and will have an important impact on cancer biology. Our ultimate goal is provide the cancer research community with (i) robust methodology to readily develop bisubstrate kinase probes for any kinase of interest and (ii) a set of highly selective kinase probes amenable to the study oncogenic signaling pathways. We will first develop a method to provide truly selective inhibitors for any kinase. Our methodology relies on a promiscuous but potent ATP-competitive fragment coupled to a highly selective peptide substrate. We will develop one bisubstrate inhibitor for each of the 9 families of the kinome. Most kinases are regulated by activation (usually via phosphorylation) and/or subcellular localization. These changes typically alter the function of the kinase and can promote oncogenesis. Herein, we will develop methodology that can convert our selective bisubstrate inhibitors into highly selective and cell-permeable `turn- on' imaging probes. These probes will be used to study the abundance and localization of active kinase across a panel of both laboratory and patient-derived TNBC cell lines. In addition, we will showcase the utility of our probes in tissue samples from TNBC biopsies.